CN114317546A - Aptamer for detecting EVD68 virus, kit and application - Google Patents

Abstract

The application discloses an aptamer for detecting EVD68 virus, a kit and application. The aptamer for detecting EVD68 virus is a nucleic acid fragment of the sequence shown in Seq ID No.1 or a complementary sequence thereof. The aptamer for detecting the EVD68 virus has high specificity and affinity, and has the advantages of small molecules, modification, easiness in synthesis, difficulty in degradation, no toxicity, no immunogenicity and the like; the problems that the EVD68 virus specific antibody is not easy to express and synthesize, is easy to degrade, has nonspecific immunogenicity and the like are solved. The aptamer for EVD68 virus detection has the advantages of high sensitivity, good specificity, wide measurement range, simplicity in operation and capability of quickly and accurately detecting EVD68 virus.

Description

Aptamer for detecting EVD68 virus, kit and application

Technical Field

The application relates to the technical field of EVD68 virus detection, in particular to an aptamer, a kit and application for detecting EVD68 virus.

Background

Enterovirus EV-D68(Enterovirus-68, EVD68 virus) is a member of the D group of enteroviruses in the small RNA virus family, has the original name rhinovirus type 87, belongs to the genus Enterovirus later, and is a single-stranded positive-sense RNA virus without an envelope. Unlike most enteroviruses, which are acid-resistant and propagate in the gastrointestinal tract, the EVD68 virus is acid-sensitive, has physical and biochemical characteristics that are greatly different from those of other enteroviruses, is rather similar to human rhinoviruses, mainly causes respiratory diseases after infection, can be manifested as nervous system diseases and acute relaxant myelitis in a few cases, and can even cause death of infected persons in severe cases. The EVD68 virus is usually epidemic in early autumn and winter, and may be slightly different according to different regions. It is susceptible to common diseases, but most of them are children and adults are recessive infections. The EVD68 virus primarily lives latent in respiratory secretions in patients who spread the virus when coughing, sneezing, or touching object surfaces. Current laboratory diagnosis of EVD68 virus infection mainly includes virus isolation, serological methods, and molecular biological methods.

Sample testing for EVD68 virus is important in clinical testing as well as in experimental studies. In the prior art, the virus infection or the detection of virus protein is mostly identified by adopting a specific antibody, and the antibody is used as a protein component and has the defects of difficult expression and synthesis, easy degradation, non-specific immunogenicity and the like; in addition, the preparation of the antibody is complex, the steps are complex, the cost is high, and certain defects exist in large-scale popularization.

Therefore, how to develop a new EVD68 virus detection technology with strong specificity and high sensitivity is a problem to be solved urgently at present.

Disclosure of Invention

The application aims to provide a novel aptamer for detecting EVD68 virus, a kit and application.

The following technical scheme is adopted in the application:

one aspect of the present application discloses an aptamer for detecting EVD68 virus, wherein the aptamer is a nucleic acid fragment of the sequence shown in Seq ID No.1 or a complementary sequence thereof.

Seq ID No.1：

5’-TTCAGCACTCCACGCATAGCTGACGTCATCCATACTGACGCACATGAGTGAGTAGACCCACCTATGCGTGCTACCGTGAA-3’。

It should be noted that the aptamer of the present application is an aptamer aiming at the epitope of the EVD68 virus target protein, which is screened by a manual screening technique, i.e., an Exponential Enrichment ligand phylogeny technique (SELEX), and has high specificity and affinity. Compared with the existing EVD68 virus specific antibody, the aptamer overcomes the problems that the antibody is not easy to express and synthesize, is easy to degrade, has nonspecific immunogenicity and the like; in addition, the aptamer has the advantages of small molecules, modification, easiness in synthesis, difficulty in degradation, no toxicity, no immunogenicity and the like. The aptamer for EVD68 virus detection has the advantages of high sensitivity, good specificity, wide measurement range and simple operation, can quickly and accurately detect the EVD68 virus target protein, is suitable for large-scale popularization, and has good market prospect. In one implementation mode of the application, the detection results of EVD68 virus infected patient samples and healthy person samples show that the positive detection rate of the aptamer to EVD68 virus infected patients, namely the sensitivity is 94%, and the positive detection rate of healthy people detection is 0%; the aptamer of the application can effectively detect EVD68 virus infected patients without false positive.

It should be noted that the key point of the present application is the research finding that the nucleic acid fragment with the sequence shown in Seq ID No.1 can be used as an aptamer for specifically detecting EVD68 virus; it will be appreciated that nucleic acid fragments of the sequence complementary to the sequence shown in Seq ID No.1 likewise have the same specificity and affinity.

In one implementation of the present application, at least one of a fluorescent substance, a nano-luminescent material, biotin, digoxigenin, and an enzyme label is bound to a nucleic acid sequence of the aptamer of the present application.

Preferably, the fluorescent species is a fluorophore, and the fluorophore comprises a FAM fluorophore or a Cy5 fluorophore. It is understood that the FAM fluorophore or Cy5 fluorophore is only one fluorophore employed in one implementation of the present application, and does not exclude that other fluorophores are also possible.

Preferably, the nano-luminescent material is a quantum dot or an up-conversion luminescent material.

Preferably, the enzyme label is horseradish peroxidase, sucrase or a functional polypeptide.

In one embodiment, the aptamer of the present invention has at least one modification selected from phosphorylation, methylation, demethylation, amination, sulfhydrylation, isotopic modification, carboxylation, thio modification, 2-methoxy modification, and inverted 3' dT/dG modification on the nucleic acid sequence.

It is important to explain that the application is to develop an aptamer capable of specifically and sensitively detecting EVD68 virus, and for specific labeling or modification of the aptamer, reference can be made to the existing aptamer labeling or modification scheme.

In another aspect of the present application, a peptide nucleic acid engineered from the aptamer of the present application is disclosed.

It should be noted that peptide nucleic acid is a kind of DNA analogue in which a polypeptide backbone is substituted for a sugar phosphate backbone, that is, a neutral peptide amide 2-aminoethylglycine bond is substituted for a pentose phosphate diester linkage backbone in DNA, and the rest is the same as DNA, and peptide nucleic acid can recognize and bind to DNA or RNA sequence in a Watson-Crick base pairing manner to form a stable double helix structure. It is understood that the peptide nucleic acids of the present application have similar specificity and sensitivity to the aptamers of the present application, since they are engineered with the aptamers of the present application.

It should be noted that the key point of the present application is to specifically detect the aptamer of the EVD68 virus, and as to how to specifically modify the aptamer into a peptide nucleic acid, reference may be made to the prior art, and no specific limitation is made herein.

In yet another aspect of the present application, a kit for detecting EVD68 virus is disclosed, the kit comprising an aptamer of the present application, or a peptide nucleic acid of the present application.

In one implementation of the present application, the kit of the present application further comprises at least one of:

(A) immobilizing a protein-coated plate of the sample solution;

(B) protein coating liquid;

(C) sealing liquid;

(D) a rinsing liquid;

(E) and (4) sample pretreatment liquid.

The key of the kit is that the kit comprises the aptamer of the application, and the aptamer can specifically detect the EVD68 virus; as for other consumables or reagents to be used, for example, (a) a protein coated plate for fixing a sample solution, (B) a protein coated solution, (C) a blocking solution, (D) a rinsing solution, and (E) a sample pretreatment solution, may be assembled into a kit as required, or may be prepared by itself or purchased separately.

In one implementation of the present application, the formulation of the protein coating solution is: 35mM NaHCO₃、13mM Na₂CO₃pH9.3-pH9.9; the formula of the sealing liquid is as follows: 136mM NaCl, 2.6mM KCl, 2mM KH₂PO₄、8mM Na₂HPO₄0.05% Tween-20, 1% BSA, pH7.1-pH7.5; the formula of the rinsing liquid is as follows: 136mM NaCl, 2.6mM KCl, 2mM KH₂PO₄、8mM Na₂HPO₄0.05% Tween-20, pH7.1-pH7.5; the formula of the sample pretreatment liquid is as follows: 150mM NaCl, 1% Triton X-100, 50mM Tris, pH7.8-pH 8.2.

In yet another aspect, the present application discloses the use of an aptamer of the present application, or a peptide nucleic acid of the present application, or a kit of the present application, in the preparation of a medicament or a tool for diagnosing or treating a disease caused by the EVD68 virus.

It is understood that the aptamers, peptide nucleic acids, and kits of the present application, are capable of specifically detecting EVD68 virus; therefore, it can also be used for diagnosing diseases caused by the EVD68 virus. For example, whether the subject has the related disease is judged by detecting whether an ex vivo sample of the subject contains the EVD68 virus or not to judge whether the subject is infected with the EVD68 virus or not. Therefore, the aptamers, peptide nucleic acids, and kits of the present application may be used to prepare drugs or tools for diagnosing or treating diseases caused by the EVD68 virus.

In yet another aspect, the present application discloses a method for detecting EVD68 virus for non-diagnostic therapeutic purposes, comprising performing EVD68 virus detection on an environment, a food product, a tool, an ex vivo sample, or a biological source material using the aptamer of the present application, or the peptide nucleic acid of the present application, or the kit of the present application.

The detection method of the present invention can be used for detecting the EVD68 virus and diseases caused by the virus; however, in addition to this, the detection of EVD68 virus in environments, food, tools, ex vivo samples, or biologically derived materials can be performed to avoid the spread or spread of EVD68 virus. For example, the EVD68 virus detection is carried out in hospitals or some special environments, and whether the environments have the risk of EVD68 virus infection or not can be judged; for another example, EVD68 virus detection of imported and exported foods, tools, ex vivo samples or biologically derived materials can avoid the input of EVD68 virus.

The beneficial effect of this application lies in:

the aptamer for detecting the EVD68 virus has high specificity and affinity, and has the advantages of small molecules, modification, easiness in synthesis, difficulty in degradation, no toxicity, no immunogenicity and the like; the problems that the EVD68 virus specific antibody is not easy to express and synthesize, is easy to degrade, has nonspecific immunogenicity and the like are solved. The aptamer for EVD68 virus detection has the advantages of high sensitivity, good specificity, wide measurement range, simplicity in operation and capability of quickly and accurately detecting EVD68 virus.

Drawings

FIG. 1 is a secondary structural diagram of an aptamer for detecting EVD68 virus in the examples of the present application.

Detailed Description

The aptamer is a single-stranded nucleic acid, generally has the size of about 20-60 bases, can form a specific three-dimensional structure through folding, and can be combined with a corresponding target molecule with high affinity and high specificity. The aptamer can be obtained by screening through an artificial screening technology (Systematic Evolution of Ligands by amplified Evolution, SELEX) simulating a natural Evolution process, and is a novel nucleic acid molecule with a recognition function. Nucleic acid aptamers with high affinity for target substances can be screened from a random single-stranded nucleic acid sequence library by using SELEX technology, and the SELEX technology is successfully applied to screening of a plurality of target substances, including metal ions, organic dyes, medicines, proteins, amino acids, various cytokines and the like. The technology has the characteristics of large library capacity, wide application range, high resolution, high affinity, relatively simple, convenient, rapid and economical screening process, high practicability and the like. The characteristics of high specificity and high affinity of the aptamer corresponding to a target substance are similar to the functions of an antibody, but the target range of the aptamer is wider, and the aptamer can be well applied to multiple aspects of medical detection, diagnosis, treatment and the like.

The reason for the high specificity and affinity of aptamers is that single-stranded nucleic acids, when encountering a target molecule, can form unique three-dimensional structures such as pockets, hairpins, G-tetramers, etc., which bind to the target molecule on a principle similar to that of antibodies specifically recognizing corresponding antigenic determinants, such as by hydrogen bonding, hydrophobic structures, ionic bonds, etc. Based on the biological characteristics of the aptamer for recognizing pathogenic microorganisms or pathological cells with high specificity, the aptamer can be widely applied to detection technology and construction of biosensors, and can realize accurate diagnosis of pathogens or diseases.

However, no aptamer for specifically detecting EVD68 virus and related research and reports thereof exist at present. Therefore, if aptamers for detecting the EVD68 virus could be developed, not only the specificity and sensitivity of the EVD68 virus detection could be improved; in addition, the problems of difficult expression and synthesis, easy degradation and non-specific immunogenicity of the antibody can be avoided.

Therefore, the SELEX technology is creatively adopted to screen the aptamer aiming at the EVD68 virus target protein epitope, so as to obtain the EVD68 virus detection aptamer with higher specificity and affinity.

Based on the above research and knowledge, the present application has developed an aptamer for detecting EVD68 virus, the aptamer being a nucleic acid fragment of the sequence shown in Seq ID No.1 or a complementary sequence thereof;

Seq ID No.1：

5’-TTCAGCACTCCACGCATAGCTGACGTCATCCATACTGACGCACATGAGTGAGTAGACCCACCTATGCGTGCTACCGTGAA-3’。

the aptamer of the present application has the following advantages:

the aptamer aiming at the EVD68 virus target protein epitope screened by the SELEX technology has higher specificity and affinity; and the preparation method has the advantages of no immunogenicity, small molecules, modification, easy synthesis, difficult degradation, no toxicity and the like. The sequence of the aptamer used in the application is obtained by screening through SELEX technology, and has complete originality.

The aptamer of the invention has high sensitivity, good specificity, wide measurement range and simple operation, can quickly detect the target protein, is suitable for large-scale popularization and has good market prospect. In one implementation of the present application, in the detection of EVD68 virus-infected patient samples and healthy human samples, the positive detection rate, i.e., the sensitivity, of EVD68 virus-infected patients is 94%; the positive detection rate of healthy people is 0%.

By adopting the aptamer of the application as a detection object, the problems of difficult expression and synthesis, easy degradation, non-specific immunogenicity and the like of an antibody can be effectively solved.

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

Example 1 aptamer for detection of EVD68 Virus

1. Determination of EVD68 virus target protein epitope

According to literature reference and bioinformatics analysis, specific epitope regions on the target protein suitable as the detection target of the aptamer are determined, and the specific epitope regions are as follows:

(1) selecting a region (amino acids 12-290) with stronger immunogenicity on the EVD68 virus VP1 protein as an aptamer screening target protein, wherein the target protein has a sequence shown as Seq ID No. 2;

Seq ID No.2：

QVESIIKTATDTVKSEINAELGVVPSLNAVETGATSNTEPEEAIQTRTVINQHGVSETLVENFLGRAALVSKKSFEYKNHASSSAGTHKNFFKWTINTKSFVQLRRKLELFTYLRFDAEITILTTVAVNGNNDSTYMGLPDLTLQAMFVPTGALTPKEQDSFHWQSGSNASVFFKISDPPARMTIPFMCINSAYSVFYDGFAGFEKNGLYGINPADTIGNLCVRIVNEHQPVGFTVTVRVYMKPKHIKAWAPRPPRTMPYMSIANANYKGRDTAPNTLN。

(2) according to the determined amino acid sequence, the nucleotide sequence is directly synthesized and prokaryotic codon optimization is carried out in a biological company, and the synthesized gene fragment is inserted into the enzyme cutting site of BamH I/Hind III of a prokaryotic expression vector pET32 a. The synthesis of nucleic acid sequence and its insertion into prokaryotic expression vector pET32a were all accomplished by Biotechnology (Shanghai) GmbH.

After obtaining the cloning vector, inducing and expressing a corresponding target protein for aptamer screening, which specifically comprises the following steps: firstly, inoculating preactivated bacterial liquid into a fresh LB culture medium according to a ratio of 1:100, and culturing at 37 ℃ for 2-2.5 h to enable the OD value of the bacterial liquid to be about 0.6-0.8; secondly, cooling the bacterial liquid in an ice-water mixture; ③ adding 0.1mM IPTG into the mixture, and inducing the mixture for 15 hours at the temperature of 16 ℃.

2. Construction of aptamer libraries and primer design

(1) In vitro synthesis of a single-stranded DNA library containing 40 random sequences, including 10, by the Gene Synthesis service of Biotechnology (Shanghai) Ltd¹⁴Individual ssDNA, i.e. random libraries of primary single-stranded DNA, nucleotide sequences of single-stranded DNAThe following were used:

5’-TTCAGCACTCCACGCATAGC-(N40)-CCTATGCGTGCTACCGTGAA-3’

among them, N40 is a single-stranded DNA of 40 random sequences.

(2) Aiming at the fixed sequence of the 5' end of the aptamer, an upstream primer P1 is designed and synthesized, wherein the upstream primer P1 is specifically a sequence shown by Seq ID No.3 and is synthesized by the company of bioengineering (Shanghai) GmbH;

Seq ID No.3：5’-TTCAGCACTCCACGCATAGC-3’。

(3) a downstream primer P2 designed and synthesized aiming at the fixed sequence of the 3 'end of the aptamer, wherein the downstream primer P2 is specifically a sequence shown by Seq ID No.4, and the 5' end of the primer is connected with a biotin group (biotin) and synthesized by biological engineering (Shanghai) corporation;

Seq ID No.4：5’-TTCACGGTAGCACGCATAGG-3’。

3. screening of nucleic acid aptamers from libraries using SELEX technology

(1) And (3) positive screening: dissolving 10nmol of initial single-stranded DNA random library in 500 mu L of PBS solution, using a constant-temperature water bath kettle at 92 ℃ for 5min, then quickly inserting the initial single-stranded DNA random library into ice for 10min in an ice bath, then incubating the treated initial single-stranded DNA random library and a target protein with the sequence shown in Seq ID No.2 on ice for 1h, then adding Ni-NTA Magnetic Agarose Beads, and continuing to incubate for 1 h. After the incubation was complete, the supernatant was removed by adsorption using a magnetic separator and the Beads were washed with 2mL of PBS. Finally, resuspending Beads with 10mL of precooled PBS, fully blowing, uniformly mixing, carrying out constant-temperature water bath at 92 ℃ for 10min, centrifuging at 13000g, and collecting supernatant, namely the single-stranded DNA library of the specific recognition target protein after the first round of screening.

(2) And (3) PCR amplification: the single-stranded DNA library that specifically recognizes the target protein after the first round of screening is subjected to PCR amplification to prepare a secondary library. The PCR reaction system is as follows: 25 mu L of Premix Tap Mix, 8 mu L of screened single-stranded DNA library, 12.5 mu L of upstream primer P and 22.5 mu L, ddH of downstream primer P₂O12. mu.L, total volume 50. mu.L. PCR cycling amplification was performed according to the following procedure: denaturation at 95 ℃ for 3min, followed by 35 cycles of amplification: 95 ℃ 30s, 55 ℃ 30s, 730s at 2 ℃, and after the circulation is finished, the reaction is stopped by extending for 10min at 72 ℃ and finally maintaining the temperature at 16 ℃. Carrying out agarose gel recovery on the library amplified by PCR to obtain a pure library fragment, wherein the recovery kit adopts

Gel Extraction Kit。

(3) Secondary single-stranded DNA library preparation: and (3) further preparing the amplified secondary library: the library fragments recovered from the agarose gel were incubated with 100. mu.L of streptavidin-labeled magnetic beads at room temperature for 20min, the double-stranded DNA library was bound to the surface of the magnetic beads by the affinity between biotin in the double-stranded DNA library and streptavidin, and then adsorbed by a magnetic separator, the supernatant was removed, and the magnetic beads were washed with 2mL of PBS. Adding a NaOH solution with a final concentration of 200mM, reacting for 10min at normal temperature to denature the double-stranded DNA library into single strands, wherein one strand with a biotin label is bonded to the magnetic beads, and the strand without the biotin label is separated from the supernatant. Collecting supernatant, removing NaOH by using a desalting column, finally adding 500 mu L of PBS, and collecting solution which is the single-stranded DNA library required by the next round of screening.

(4) Repeating the steps of (1) positive screening, (2) PCR amplification and (3) secondary single-stranded DNA library preparation for 15 times, wherein the single-stranded DNA library obtained by screening has the strongest identification capability on the target protein.

The single-stranded DNA obtained by the final screening is sent to the department of biotechnology, Inc. for sequencing.

The sequencing result shows that the single-stranded DNA finally screened in the example is the sequence shown in Seq ID No.1, namely the aptamer for detecting the EVD68 virus in the example.

Seq ID No.1：

5’-TTCAGCACTCCACGCATAGCTGACGTCATCCATACTGACGCACATGAGTGAGTAGACCCACCTATGCGTGCTACCGTGAA-3’。

(5) In order to sensitively detect the reaction product in the reaction system, the 5' end of the aptamer having the sequence shown in Seq ID No.1 is labeled with FAM fluorophore. Aptamers carrying FAM fluorophore labels were synthesized by Biotechnology (Shanghai) Inc.

Example 2 verification of aptamers

1. Secondary structure prediction

In this example, RNA Structure Programming software was used to perform secondary Structure prediction of the aptamer having the sequence shown in Seq ID No.1, which was obtained by screening in example 1, as shown in FIG. 1.

The results in FIG. 1 show that aptamers of the sequence shown in Seq ID No.1 can form specific stem-loop structures and hairpin structures.

2. Binding assays for aptamers to target proteins

The ForteBio Octet RED96 interaction analyzer was used to detect the binding force of aptamers to target proteins and PBS was set as a control, and the experimental principles and procedures were referenced in the following web pages:

http://hzaml.hzau.edu.cn/info/1107/3266.htm#/

the results of the ForteBio Octet RED96 interaction analyzer are shown in Table 1.

TABLE 1 binding force test results of aptamers to target proteins

Aptamer	Dissociation constant Kd (nM) to target protein
		EVD68 Virus-aptamer	8.0
PBS control	Without bonding

The results in Table 1 show that aptamers having the sequence shown in Seq ID No.1 bind specifically to VP1 protein of EVD 68.

3. Specificity detection

In the present embodiment, human serum albumin, immune serum globulin, EVD68 virus VP1 protein and aptamer are respectively used for specific detection, and the following web pages can be referred to for the experimental principle and the operation steps:

http://hzaml.hzau.edu.cn/info/1107/3266.htm#/

the experimental result shows that the aptamer in example 1 has no combination with human serum albumin and immune serum globulin, and only combines with the VP1 protein of the EVD68 virus; it is demonstrated that the aptamer of example 1 has better specificity.

4. Stability test

At room temperature, 0.2. mu.g of the aptamer of example 1 was taken and put in 1mL of serum, and 0.2. mu.g of the aptamer of example 1 was taken and put in an aqueous solution. After standing at room temperature for 4 weeks, detection was carried out by RT-PCR. The upstream primer P1 is the sequence shown in Seq ID No.3, and the downstream primer P2 is the sequence shown in Seq ID No.4, the PCR system is as follows: 25 μ L of Premix Tap Mix, 8 μ L of aptamer dissolved in serum or aqueous solution, 12.5 μ L of upstream primer P, and 22.5 μ L, ddH of downstream primer P₂O12. mu.L, total volume 50. mu.L. PCR cycling amplification was performed according to the following procedure: denaturation at 95 ℃ for 3min, followed by 35 cycles of amplification: 30s at 95 ℃, 30s at 55 ℃ and 30s at 72 ℃, after the circulation is finished, the extension is carried out for 10min at 72 ℃, and finally the reaction is stopped by maintaining the temperature at 16 ℃.

The detection result shows that the aptamer placed at normal temperature has a stable structure and is not degraded.

5. Experiment of viral infection

Sensitive host cells were infected with EVD68 virus, while a negative control group without infection was set up. Cells were plated in 6-well plates, infected at an MOI of 0.1, and cultured in a cell incubator at 37 ℃ for 36 h. After 36h of infection, the cells were trypsinized, 200. mu.L of pancreatin was added to each well, centrifuged at 2000rpm to obtain cell pellets and the supernatant was removed and resuspended in 500. mu.L of PBS. Fixing and permeabilizing the cells, specifically referring to webpage https:// www.novopro.cn/articles/201606271111.html #/; and mixing the aptamer which corresponds to the EVD68 virus and is provided with the fluorescent group label with the cell suspension infected with the virus and the cell suspension of the uninfected control group respectively for 1h to ensure that the final concentration reaches about 1 mu g/mL, and analyzing and calculating the fluorescence value of the FAM channel by adopting a flow cytometer.

The result shows that the fluorescence value of the aptamer label corresponding to the cell sample infected with the EVD68 virus is remarkably increased, and the aptamer obtained by screening in example 1 can be used for detecting the EVD68 virus infection.

Example 3 kit for detecting EVD68 Virus

The kit for detecting the EVD68 virus of this example contains the following formulation:

(A) immobilizing a protein-coated plate of the sample solution; in this example, 96-well protein coated plates were used;

(B) protein coating liquid;

(C) sealing liquid;

(D) a rinsing liquid;

(E) sample pretreatment liquid;

(F) example 1 selected aptamers carrying a FAM fluorophore label.

Wherein, (a) a 96-well protein-coated plate for immobilizing a sample solution: the commercially available ELISA plate having protein adsorption ability was used as it was. (B) Protein coating liquid: belongs to a conventional molecular detection reagent, and the formula is 35mM NaHCO₃、13mM Na₂CO₃pH9.3-pH9.9, prepared with double distilled water. (C) Sealing liquid: belongs to a conventional molecular detection reagent, and the formula is 136mM NaCl, 2.6mM KCl and 2mM KH₂PO₄、8mM Na₂HPO₄0.05% Tween-20, 1% BSA, pH7.1-pH7.5, prepared using double distilled water. (D) Rinsing liquid: belongs to a conventional molecular detection reagent, and the formula is 136mM NaCl, 2.6mM KCl and 2mM KH₂PO₄、8mM Na₂HPO₄0.05% Tween-20, pH7.1-pH7.5, prepared using double distilled water. (E) Sample pretreatment liquid: belongs to a conventional molecular detection reagent, and the formula of the kit is 150mM NaCl, 1% Triton X-100 and 50mM Tris, and the pH value is 7.8-8.2. (F) Aptamer with fluorophore label: examples of the invention1 the synthesized aptamer with FAM fluorophore label is dissolved in TE buffer solution, and the working concentration is 200nM when using.

EXAMPLE 4 use of the kit

A virus infection experiment was performed using the kit of example 4 using 50 clinical pharyngeal swab samples for which positive EVD68 virus had been confirmed and 50 clinical pharyngeal swab samples for which no EVD68 virus had been confirmed as a control.

Meanwhile, three commercially available EVD68 virus antigen detection kits were used as controls, and three commercially available EVD68 virus antigen detection kits were labeled as kit a, kit B, and kit C, respectively.

The specific experimental method comprises the following steps:

1. all reagents and samples were allowed to return to room temperature before use, and the samples to be tested were diluted with the coating solution.

2. To the corresponding wells, 100. mu.L of the sample to be tested was added and incubated at room temperature for 2 hours. The coating time can be optimized and adjusted according to the actual situation.

3. Discard solution and wash 2 times with rinse, each time adding 200. mu.L of rinse to the sample well. After the last wash is completed, the residual rinse liquid is removed by suction or pouring.

4. Add 200. mu.L of blocking solution to the sample wells and incubate for 2 hours at room temperature.

5. The solution was discarded. Repeating the washing step of step 3.

6. Add 100. mu.L of aptamer with fluorophore label to the sample wells and incubate for 2 hours at room temperature in the dark. The incubation time can be optimally adjusted according to the actually detected fluorescence signal intensity.

7. The solution was discarded. The washing step of step 3 was repeated for a total of 4 washes.

8. Detecting the sample hole by using a full-band laser microplate reader, and setting as follows: the excitation wavelength is 494nm, and the detection wavelength is 522 nm.

The result judgment mode is as follows: a fluorescence value greater than 50 indicates a positive result.

The test results of the test kit of example 4 and the test kits a, B and C as controls were counted for 50 EVD68 virus positive samples and 50 EVD68 virus-free control samples, and the results are shown in table 2.

TABLE 2 detection results of the aptamer and the antigen detection kit for positive and control samples

	Nucleic acid aptamers	Kit A	Kit B	Kit C
					EVD68 Virus-positive sample	47/50	45/50	46/50	45/50
Control sample without EVD68 Virus	0/50	1/50	1/50	0/50

The data in table 2 show the number of positive results and samples, for example, "47/50" indicates that 47 positive results were detected in 50 EVD68 virus positive samples, and "0/50" indicates that 0 positive results were detected in 50 control samples without EVD68 virus.

The results in table 2 show that the kit of example 4 can effectively detect an EVD68 virus positive sample with a sensitivity of 94%, while no false positive was detected for the control sample without EVD68 virus; the sensitivity of the kit A, the kit B and the kit C to EVD68 virus positive samples is lower than that of the aptamer screened in example 1, and the kit A and the kit B respectively have false positive detection.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.

SEQUENCE LISTING

<110> Shitong lan Dada (Shenzhen) Biotech development Limited

<120> aptamer for detecting EVD68 virus, kit and application

<130> 21I33069

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 80

<212> DNA

<213> EVD68 Virus aptamer

<400> 1

ttcagcactc cacgcatagc tgacgtcatc catactgacg cacatgagtg agtagaccca 60

cctatgcgtg ctaccgtgaa 80

<210> 2

<211> 279

<212> PRT

<213> immunogenic region of VP1 protein of EVD68 virus

<400> 2

Gln Val Glu Ser Ile Ile Lys Thr Ala Thr Asp Thr Val Lys Ser Glu

1 5 10 15

Ile Asn Ala Glu Leu Gly Val Val Pro Ser Leu Asn Ala Val Glu Thr

20 25 30

Gly Ala Thr Ser Asn Thr Glu Pro Glu Glu Ala Ile Gln Thr Arg Thr

35 40 45

Val Ile Asn Gln His Gly Val Ser Glu Thr Leu Val Glu Asn Phe Leu

50 55 60

Gly Arg Ala Ala Leu Val Ser Lys Lys Ser Phe Glu Tyr Lys Asn His

65 70 75 80

Ala Ser Ser Ser Ala Gly Thr His Lys Asn Phe Phe Lys Trp Thr Ile

85 90 95

Asn Thr Lys Ser Phe Val Gln Leu Arg Arg Lys Leu Glu Leu Phe Thr

100 105 110

Tyr Leu Arg Phe Asp Ala Glu Ile Thr Ile Leu Thr Thr Val Ala Val

115 120 125

Asn Gly Asn Asn Asp Ser Thr Tyr Met Gly Leu Pro Asp Leu Thr Leu

130 135 140

Gln Ala Met Phe Val Pro Thr Gly Ala Leu Thr Pro Lys Glu Gln Asp

145 150 155 160

Ser Phe His Trp Gln Ser Gly Ser Asn Ala Ser Val Phe Phe Lys Ile

165 170 175

Ser Asp Pro Pro Ala Arg Met Thr Ile Pro Phe Met Cys Ile Asn Ser

180 185 190

Ala Tyr Ser Val Phe Tyr Asp Gly Phe Ala Gly Phe Glu Lys Asn Gly

195 200 205

Leu Tyr Gly Ile Asn Pro Ala Asp Thr Ile Gly Asn Leu Cys Val Arg

210 215 220

Ile Val Asn Glu His Gln Pro Val Gly Phe Thr Val Thr Val Arg Val

225 230 235 240

Tyr Met Lys Pro Lys His Ile Lys Ala Trp Ala Pro Arg Pro Pro Arg

245 250 255

Thr Met Pro Tyr Met Ser Ile Ala Asn Ala Asn Tyr Lys Gly Arg Asp

260 265 270

Thr Ala Pro Asn Thr Leu Asn

275

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<400> 3

ttcagcactc cacgcatagc 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<400> 4

ttcacggtag cacgcatagg 20

Claims (10)

1. An aptamer for detecting EVD68 virus, comprising: the aptamer is a nucleic acid fragment of a sequence shown in Seq ID No.1 or a complementary sequence thereof;

Seq ID No.1：

5’-TTCAGCACTCCACGCATAGCTGACGTCATCCATACTGACGCACATGAGTGAGTAGACCCACCTATGCGTGCTACCGTGAA-3’。

2. the aptamer according to claim 1, wherein: at least one of fluorescent substance, nano luminescent material, biotin, digoxigenin and enzyme label is combined on the nucleic acid sequence of the aptamer.

3. The aptamer according to claim 2, wherein: the fluorescent substance is a fluorophore, and the fluorophore comprises a FAM fluorophore or a Cy5 fluorophore;

the nano luminescent material is quantum dot or up-conversion luminescent material;

the enzyme label is horseradish peroxidase, sucrase or functional polypeptide.

4. The aptamer according to any one of claims 1 to 3, wherein: the nucleic acid sequence of the aptamer has at least one modification of phosphorylation, methylation, demethylation, amination, sulfhydrylation, isotopic ization, carboxylation, sulfo modification, 2-methoxyl modification and 3' end inversion dT/dG modification.

5. A peptide nucleic acid modified from the aptamer according to any one of claims 1 to 4.

6. A kit for detecting EVD68 virus, comprising: the kit comprises the aptamer according to any one of claims 1 to 4, or the peptide nucleic acid according to claim 5.

7. The kit of claim 6, wherein: also included is at least one of the following,

(A) immobilizing a protein-coated plate of the sample solution;

(B) protein coating liquid;

(C) sealing liquid;

(D) a rinsing liquid;

(E) and (4) sample pretreatment liquid.

8. The kit of claim 7, wherein: the formulation of the protein coating solution is 35mM NaHCO₃、13mM Na₂CO₃，pH9.3-pH9.9；

The formula of the confining liquid is 136mM NaCl, 2.6mM KCl and 2mM KH₂PO₄、8mM Na₂HPO₄、0.05％Tween-20、1％BSA，pH7.1-pH7.5；

The formula of the rinsing liquid is 136mM NaCl, 2.6mM KCl and 2mM KH₂PO₄、8mM Na₂HPO₄、0.05％Tween-20，pH7.1-pH7.5；

The formula of the sample pretreatment liquid is 150mM NaCl, 1% Triton X-100, 50mM Tris, pH7.8-pH8.2.

9. Use of the aptamer according to any one of claims 1 to 4, or the peptide nucleic acid according to claim 5, or the kit according to any one of claims 6 to 8, for the preparation of a medicament or a tool for the diagnosis or treatment of diseases caused by the EVD68 virus.

10. A method for detecting EVD68 virus for non-diagnostic therapeutic purposes, comprising: comprising performing an EVD68 virus assay on an environment, a food product, a tool, an ex vivo sample, or a biologically derived material using the aptamer of any one of claims 1 to 4, or the peptide nucleic acid of claim 5, or the kit of any one of claims 6 to 8.

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